The goal of this application is to develop Dr. John Lee into an independent physician/scientist. Drs. Welsh and Klingelhutz will assume responsibility as mentors to ensure success in his development. The heart of this proposal is intensive laboratory training in the pathogenesis of human papilloma virus (HPV) related airway disease. HPV infection of the aerodigestive tract results in papilloma formation and the development of squamous cell cancer, leading causes of dysphonia and aphonia. An understanding of disease pathogenesis has been limited by the lack of an in vitro model of differentiated human airway epithelia (HAE) and the inability to produce experimentally useful quantities of HPV virions. However, our lab now routinely produces in vitro models of well-differentiated HAE. Moreover, studies in keratinocytes have circumvented the lack of HPV virions by delivering the HPV genome by transfection. However, airway epithelia have marked differences from keratinocytes, and comparable transfection methods cannot be used in airway epithelia. Therefore, I developed a novel strategy to deliver an HPV genome inserted in a recombinant adenovirus. In preliminary studies, I demonstrated the feasibility of this approach by transferring the HPV genome with high efficiency to well-differentiated HAE. The HPV genome was excised from the vector and circularized. These results allow me to now ask the following questions about the interaction between HPV and HAE. 1) What factors influence the establishment of viral persistence in human airway epithelia? I will learn whether the HPV genome persists in HAE, and whether it exists as an episome or as integrated DNA. The data will also show us how epithelial differentiation influences the pattern of genome persistence. 2) Does the HPV genome alter the morphology or differentiation of human airway epithelia? Studies addressing this question will help us understand cellular changes associated with HPV-induced alterations of the epithelium in vivo. 3) Do human airway epithelia produce infectious HPV virions? The answer to this question will provide important insight into the factors involved in a productive viral infection. 4) What is the temporal and spatial pattern of HPV gene expression in differentiated human airway epithelia? An investigation of the timing, localization, and quantity of viral mRNA and protein expression will begin to show us how differentiation impacts the viral life cycle. These studies will provide new insight into HPV-related airway disease and should hasten the development of new treatments.